The development of multicellular organisms requires that specific fates be assigned to cells and that cells "memorize" these instructions through many cycles of cell division. A well documented example involves the transcriptional regulation of the Drosophila Antennapedia (ANT-C) and bithorax (BX-C) gene complexes. The initial patterns of ANT-C and BX-C expression are controlled by transcription factors that are encoded by the segmentation genes. However, shortly after these patterns are established, the products of the segmentation genes are degraded. Maintenance of ANT-C and BX-C transcriptional activity and repression then becomes the respective responsibilities of the trithorax-group (trx-G) and Polycomb-group (Pc-G) proteins. Homologs of trx-G and Pc-G genes have been identified in a wide variety of organisms and mutations in some are associated with oncogenesis. Our long-term goal is to understand the molecular mechanisms by which the Pc-G and trx-G maintain the transcriptional states of target genes. The experiments described in this proposal are designed to better define the molecular activities of one Pc-G protein, Enhancer of zeste [E(z)]. In addition to its role in Pc-G mediated repression, E(z) may also participate in trx-G-mediated activation. E(z) protein has been shown to interact two other Pc-G proteins, and another protein that shares extensive sequence similarity with a human protein that is a component of repression complexes. Preliminary evidence suggests that several trx- G proteins are also potential E(z) binding partners. All of these interactions will be analyzed using yeast two-hybrid, in vitro binding, and co-immunoprecipitation assays. The in vivo functions of these specific protein-protein relationships will be defined through genetic analysis of E(z) transgenes bearing point mutations that specifically disrupt the respective interactions. The in vivo associations of E(z) with these and other proteins also will be examined by characterizing native E(z)-containing complexes from embryo extracts.